He Archaeal domain. The multiple alignment of the ORF sequences from P. Autophagy furiosus, P. horikoshii, P. abyssi, and T. kodakarensis is shown in Fig. 8. The sequence identities among these proteins are 70 on average. The newly discovered nuclease likely functions in DNA repair in Thermococcales lineage. It would be interesting if these organisms have a specific DNA repair system including this protein for the DNA damages occurring due to the high temperatures of their habitat.Comparison of the Binding Affinities of PfuExo I to Various DNA SubstratesTo further characterize PfuExo I, gel mobility shift assays were performed using different types 
of DNA in the absence of Mg2+, to prevent substrate loss due to degradation, as shown in Fig. 7. The shifted bands should correspond to the DNA-PfuExo I complexes. Multiple shifted bands were observed with the ssDNA substrate (Fig. 7A). As slower migrating complexes were observed with increasing protein concentrations, PfuExo I may bind ssDNA nonspecifically at higher concentrations. Slight shifts were observed when dsDNA was used (Fig. 7B). Shifted bands were distinctly observed from both the 39- and 59-overhang DNAs, butIdentification of Novel Nuclease from P. furiosusFigure 6. Endonuclease activity of PfuExo I. Purified PfuExo I was incubated with circular ssDNA (M13 mp18) or dsDNA (pBR322) in the reaction condition described in Materials and Methods. Reaction products were analyzed by electrophoresis on a 1 agarose gel, followed by ethidium bromide staining. doi:10.1371/journal.pone.0058497.gFigure 5. Cleavage specificity of PfuExo I. Purified PfuExo I (10 nM) was incubated with 59- overhang and 39-overhang DNAs (5 nM) for various times at 55uC. The positions labeled by 32P are indicated by an asterisk for each substrate. Epigenetics Aliquots were removed from the reactions, quenched, and then resolved by PAGE on a 12 gel containing 8 M urea. doi:10.1371/journal.pone.0058497.gDiscussionArchaea are unique organisms that are evolutionally different from bacterial and eukaryotic organisms. To understand their DNA repair systems, several archaeal proteins homologous to the eukaryotic and bacterial proteins involved in the DNA repair processes have been identified and biochemically analyzed to date. The hyperthermophilic archaea should have especially efficient DNA repair systems, due to their habitation in extreme environments. However, the DNA repair system in Archaea is still not well understood, and many more efforts are required to determine the similarities and differences between the archaeal DNA repair system and those of Bacteria and Eukarya. For example, the thermophilic archaea lack the NER damagerecognition proteins (eukaryotic XPA and XPC or bacterial UvrA and UvrB) and the MMR-specific proteins (MutS and MutL) [25]. Every factor that seems to be involved in DNA repair systems must be identified to elucidate the molecular mechanisms of damaged DNA repair in Archaea, and the exonucleases definitely play important roles in the processes. It would be especially interesting to determine whether any unique repair ability is present in the third domain of life. In E. coli cells, several proteins function as single-stranded DNA specific exonucleases. RecJ, Exo I, Exo VII, and Exo X are involved in MMR and HR. In Archaea, a 59-39 exonuclease, NurA, is conserved only in the thermophilic archaea, and the nurA gene is organized in an operon structure with rad50 and mre11, which are involved in double-stranded.He Archaeal domain. The multiple alignment of the ORF sequences from P. furiosus, P. horikoshii, P. abyssi, and T. kodakarensis is shown in Fig. 8. The sequence identities among these proteins are 70 on average. The newly discovered nuclease likely functions in DNA repair in Thermococcales lineage. It would be interesting if these organisms have a specific DNA repair system including this protein for the DNA damages occurring due to the high temperatures of their habitat.Comparison of the Binding Affinities of PfuExo I to Various DNA SubstratesTo further characterize PfuExo I, gel mobility shift assays were performed using different types of DNA in the absence of Mg2+, to prevent substrate loss due to degradation, as shown in Fig. 7. The shifted bands should correspond to the DNA-PfuExo I complexes. Multiple shifted bands were observed with the ssDNA substrate (Fig. 7A). As slower migrating complexes were observed with increasing protein concentrations, PfuExo I may bind ssDNA nonspecifically at higher concentrations. Slight shifts were observed when dsDNA was used (Fig. 7B). Shifted bands were distinctly observed from both the 39- and 59-overhang DNAs, butIdentification of Novel Nuclease from P. furiosusFigure 6. Endonuclease activity of PfuExo I. Purified PfuExo I was incubated with circular ssDNA (M13 mp18) or dsDNA (pBR322) in the reaction condition described in Materials and Methods. Reaction products were analyzed by electrophoresis on a 1 agarose gel, followed by ethidium 
bromide staining. doi:10.1371/journal.pone.0058497.gFigure 5. Cleavage specificity of PfuExo I. Purified PfuExo I (10 nM) was incubated with 59- overhang and 39-overhang DNAs (5 nM) for various times at 55uC. The positions labeled by 32P are indicated by an asterisk for each substrate. Aliquots were removed from the reactions, quenched, and then resolved by PAGE on a 12 gel containing 8 M urea. doi:10.1371/journal.pone.0058497.gDiscussionArchaea are unique organisms that are evolutionally different from bacterial and eukaryotic organisms. To understand their DNA repair systems, several archaeal proteins homologous to the eukaryotic and bacterial proteins involved in the DNA repair processes have been identified and biochemically analyzed to date. The hyperthermophilic archaea should have especially efficient DNA repair systems, due to their habitation in extreme environments. However, the DNA repair system in Archaea is still not well understood, and many more efforts are required to determine the similarities and differences between the archaeal DNA repair system and those of Bacteria and Eukarya. For example, the thermophilic archaea lack the NER damagerecognition proteins (eukaryotic XPA and XPC or bacterial UvrA and UvrB) and the MMR-specific proteins (MutS and MutL) [25]. Every factor that seems to be involved in DNA repair systems must be identified to elucidate the molecular mechanisms of damaged DNA repair in Archaea, and the exonucleases definitely play important roles in the processes. It would be especially interesting to determine whether any unique repair ability is present in the third domain of life. In E. coli cells, several proteins function as single-stranded DNA specific exonucleases. RecJ, Exo I, Exo VII, and Exo X are involved in MMR and HR. In Archaea, a 59-39 exonuclease, NurA, is conserved only in the thermophilic archaea, and the nurA gene is organized in an operon structure with rad50 and mre11, which are involved in double-stranded.